Project Summary/Abstract The ability of the cell to tightly regulate the temporal and spatial movement of molecules across membranes is central to its survival. This movement has to be done in a selective manner to ensure that the chemistry of the cytoplasm and other internal compartments are not disturbed. To carry out these tasks, membranes contain transporters and channels that are often specific to particular cell types or organelles. The primary objective of the current proposal is to use computational methods to examine the conformational changes and functional operation of the SGLT sugar cotransporters and the closely related sialic acid transporter nanT. Most of our efforts are focused on vSGLT, the bacterial member of the solute sodium symporter family of cotransporters, whose human homologues are responsible for adsorption of simple sugars in the small intestine and kidneys. vSGLT is related to a very large superfamily of transporters called the Leucine Transporter (LeuT) superfamily, which include serotonin transporters, sodium iodide transporters and other important pharmacological targets. An increased understanding of the molecular workings of these transporters has the potential to help in treating diseases related to type 2 diabetes mellitus (T2DM), severe dehydration, and depression. In Aim 1, we will study how Na+ and substrate bind to the outward-facing state of cotransporters, an important first step in recognition and entry into the cell. We hypothesize that Na+ binds first to prime the protein for substrate binding. We will take advantage of our collaborator's recent determination of the high-resolution structure of the sialic acid transporter (nanT) at 2.0 in the outward-facing state. We will then compute the energetics of outer gate closing. We posit that cargo loading will help stabilize the gate in a closed conformation. Next, we will create an outward-facing model of vSGLT based on nanT and validate the model with experiments in the Abramson lab (UCLA) such as DEER distance measurements (with Mchaourab lab, Vanderbilt), WAXS studies (with Neutze lab, Gothenburg), and uptake assays. Extracellular sugar and Na+ binding will then be studied using computation. Our goal in Aim 2 is to use computational drug design to reveal the structural basis of inhibitor binding to human SGLT2 (a T2DM target) and find small molecules that bind vSGLT in a conformationally selective manner. Our efforts on hSGLT2 will be coupled with screening in the Wright lab (UCLA), which could lead to improved T2DM therapies. Meanwhile, small molecules that bind vSGLT in distinct states, which do not exist, would provide tools for stabilizing and crystallizing the unknown, outward-facing structure of vSGLT as well as interpreting spectroscopic data. In Aim 3, we will use enhanced sampling methods (such as the Weighted Ensemble method or Markov State Modeling) to simulate the entire transport cycle and reveal how Na+ and substrate drive the cotransporter through key conformational states ? thus revealing the mechanistic underpinnings of membrane transport in this important superfamily.